TY - JOUR
T1 - Development of a Double-Crossover Markerless Gene Deletion System in <span class="named-content genus-species" id="named-content-1">Bifidobacterium longum</span>: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation
JF - Applied and Environmental Microbiology
JO - Appl. Environ. Microbiol.
SP - 4984
LP - 4994
DO - 10.1128/AEM.00588-12
VL - 78
IS - 14
AU - Hirayama, Yosuke
AU - Sakanaka, Mikiyasu
AU - Fukuma, Hidenori
AU - Murayama, Hiroki
AU - Kano, Yasunobu
AU - Fukiya, Satoru
AU - Yokota, Atsushi
Y1 - 2012/07/15
UR - http://aem.asm.org/content/78/14/4984.abstract
N2 - Functional analysis of Bifidobacterium genes is essential for understanding host-Bifidobacterium interactions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA, which lacks the plasmid replication gene repA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors repA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in B. longum 105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using the aga gene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of aga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-complemented strain. These functional analyses revealed that aga is the only gene encoding a functional α-galactosidase enzyme in B. longum 105-A.
ER -